pXa-1 plasmid can be obtained from Promega (Madison, WI).
pDisplay vector, lipofectamine, and primers 5'-ctcgtcagatcttcgaaactgaaggtaaca-3' (upstream) and 5'-atactcccgcggggaaccttcgatgagctcg-3' (downstream) as well as DH10B electrocompetent cells can be purchased from Invitrogen (Carlsbad, CA).
Bgl II, Sac II restriction enzymes and their reaction buffer (10x buffer 2), T4 DNA ligase and its reaction buffer (10x ligase buffer) as well as 1kb DNA ladder can be obtained from New England Biolabs (Beverly, MA).
Quiagen gel purification, Miniprep and Maxiprep kits can be obtained from Qiagen (Valencia, CA).
Protease inhibitor, anti-HA antibody can be purchased from Roche (Indianapolis, IN).
Bradford reagent and 10% polyacrylamide minigels can be obtained from BIO-RAD (Hercules, CA).
ECL-Anti-mouse antibody-horse radish peroxidase (HRP) conjugate (NA931V) can be obtained from Amersham Biosciences (Piscataway, N.J).
Anti-mouse-Alexa594, Alexa660 and Alexa680 conjugates, anti-biotin antibody and streptavidin-HRP can be obtained from Molecular Probes (Eugene, OR).
ECL Reagent and SuperBlock buffer can be purchased from Pierce Biotech (Rockford, IL).
Fluorescent mounting medium can be obtained from DakoCytomation (Carpinteria, CA).
Poly-lysine, sodium chloride, ampicillin, Ponseau staining solution, paraformaldehyde (PFA) and ethidium bromide can be purchased from Sigma (St. Louis, MO).
Purina Biotin deficient diet 5836 can be obtained from Purina Test Diet (Richmond, IN).
Thermostable high fidelity Pfu DNA polymerase and its buffer can be obtained from Stratagene (La Jolla, CA).
Agarose can be purchased from ISCBioExpress (Kaysville, UT).
Fetal bovine serum (FBS), penicillin and streptomycin mix, Dulbecco's modified Eagle's medium (DMEM) and OPTIMEM, 10x phosphate buffere saline (PBS) can be obtained from cellgro by Mediatech Inc. (Herndon, VA).
Bacto-tryptone, Bacto-yeast extract and Baco-Agar can be obtained from Becton Dickinson and Company (Sparks, MD).
Tissue culture plates, microscope cover glass and sodium dodecyl sulfate (SDS) can be obtained from Fisher Scientific (Pittsburg, PA)
Cell lysis buffer: 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate in 50 mM Tris-HCl, pH 8. store at 4 0C. Make fresh every 1-2 weeks.
50x TAE buffer: 2 M Tris-acetate, 50 mM EDTA (pH 8.0). to prepare 1 L add 242 g Tris base, 57.1 ml glacial acetic acid and 100 ml 0.5M EDTA, adjust to pH 8.0. Store at room temperature.
6x Gel loading buffer: 30% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol. Store at room temperature.
4x polyacrylamide loading buffer: to make 8mL, 4mL Tris-HCl (0.5M), pH 6.8, 1.6 mL 20% SDS, 5mg Bromophenol blue, 1.6 mL glycerol, 0.8 mL beta-mercaptoehanol. Store at -20 C.
10x Running buffer: to make 1L, add 30.2g Tris, 144 g Glycine, 10 g of SDS and increase the volume with ddH2O.
10x Transfer buffer: To prepare 1 L, add 29 g Tris, 147 g Glycine and increase the volume with ddH2O
10x TBST: To prepare 1L, 80 g of NaCl, 30 g Tris, pH 8, add 5 mL of Tween-20 and increase the volume with ddH2O.
SOB medium: To make 1L, 20 g bacto-tryptone, 5 g bacto-yeast extract, 0.5 g NaCl and increase the volume with ddH2O. Sterilize by autoclaving for 20-30 min.
SOC medium: To SOB medium add 20 mM of sterile glucose and store at -20 C. 1M solution of glucose is prepared by dissolving 18 g of Glucose in 100 mL of ddH2O and is sterilized by filtration through 0.22-micron filter.
Luria-Bertanu (LB) medium. To make 1 L, add 10g bacto-tryptone, 5g bacto-yeast extract, 10g NaCl and increase the volume with ddH2O). Mix well and sterilize by autoclaving as above.
LB-agar plates with ampicillin: To 1 L of LB medium, add 15 g of agar and sterilize as above. After cooling to around 60 C, add 500 µl of 100 mg/ml ampicillin (prepared in ddH2O and stored at -20 C) and poor it into plates near flame. Once solidified, seal plates with parafilm and store at 4 C. Make fresh every one month.